Isolated phosphorylcholine binding lymphocytes. I. Use of a cleavable crosslinking reagent for solid-phase adsorbent isolation of functional antigen binding cells.
Isolation and characterization of BALB/c lymphocytes specific for phosphorylcholine is described. The isolation protocol utilizes phosphorylcholine coupled to gelatin coated plates via the cleavable crosslinking reagent N-succinimidyl 3-(2-pyridyldiothio)propionate (SPDP). The procedure is rapid, requiring only 1-2 h and conducted entirely at 0-4 degrees C. Hapten binding cells are eluted by vigorous pipetting at 4 degrees C with medium containing 20% fetal calf serum. Approximately 70% of isolated cells rebind antigen as assessed using a PC Brucella abortus rosette assay while 50% express the TEPC15 idiotype. Approximately 85% of isolated and idiotype positive cells are B cells while the remainder are T cells. Limiting dilution analysis revealed that approximately 1/5 of PC binding cells isolated from the spleens of normal mice respond to lipopolysaccharide plus dextran sulfate by production of anti-PC antibody. Approximately 1/11 respond to PC Brucella abortus and 1/250 respond to PC sheep erythrocytes plus primed T cells by anti-PC antibody production. The results clearly demonstrate the effectiveness of this technique for isolation of highly enriched, functional, antigen specific lymphocytes.
D000818:Animals; D000921:Antibody-Producing Cells / Q000166:cytology / Q000378:metabolism; D001665:Binding Sites; D002003:Brucella abortus / Q000378:metabolism; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002794:Choline / Q000031:analogs & derivatives*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006736:Horses; D006801:Humans; D007163:Immunosorbent Techniques; D008214:Lymphocytes / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010767:Phosphorylcholine / Q000276:immunology / Q000378:metabolism*; D010954:Plasmacytoma / Q000276:immunology; D011817:Rabbits; D012397:Rosette Formation; D013154:Spleen / Q000166:cytology; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D013388:Succinimides; D010767:Phosphorylcholine; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; D002794:Choline